Polypeptide capable of reacting with antibodies of patients suffering from multiple sclerosis and uses

ABSTRACT

The invention concerns a polypeptide specifically reacting with the antibodies of patients suffering from multiple sclerosis (SEP) and whereof the peptide sequence comprises at least one sequence selected among SEQ ID No. 1 to SEQ ID NO: 19, and their equivalent sequences, and the use of this polypeptide in a reagent and a kit for detecting multiple sclerosis, an immunoreactive composition and in a method for fixing, in a biological sample, antibodies characteristic and/or specific of multiple sclerosis.

The present invention relates to the determination of immunoreactivepolypeptides capable of reacting specifically with the antibodies ofpatients suffering from multiple sclerosis (MS), and the use of thesepolypeptides.

The Applicant has defined a polypeptide capable of reacting specificallywith the antibodies of patients suffering from multiple sclerosis, andwhich must additionally meet at least any one of the followingdefinitions, provided that said polypeptide is different from thepolypeptides having one of the following sequences: SEQ ID NO: 20, SEQID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25,SEQ ID NO: 26, SEQ ID NO: 27:

its peptide sequence comprises at least one sequence chosen from SEQ IDNO: 1 to SEQ ID NO: 19, and their equivalent sequences;

its peptide sequence consists of a sequence chosen from SEQ ID NO: 1 toSEQ ID NO: 19, and their equivalent sequences; preferably, it is chosenfrom SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ IDNO: 16 and SEQ ID NO: 19;

it comprises a sequence equivalent to SEQ ID NO: 11, said equivalentsequence exhibiting, for a succession of 8 contiguous amino acids, atleast 35% (which corresponds to at least 3 amino acids), preferably 50%(which corresponds to at least 4 amino acids) identity, and/or at least60% (which corresponds to about at least 5 amino acids), preferably 75%(which corresponds to at least 6 amino acids) homology with a sequenceof the foot-and-mouth disease virus SAT3 protein, said polypeptide beingdifferent from the whole or part of said SAT3 protein;

it comprises a sequence equivalent to SEQ ID NO: 13 exhibiting, for asuccession of 12 contiguous amino acids, at least 40%, preferably 50%identity, and/or at least 55%, preferably 65% homology with a sequencep30/p10/5′v-fsm of the coding region of the feline sarcoma virus (FSV)[NCBI reference gi/554646], said polypeptide being different from thewhole or part of said sequence p30/p10/5′v-fsm.

Moreover, the work by the Applicant, in the search for an etiology ofMS, has led to the discovery of the existence of at least onepathological and/or infective agent, the retrovirus MSRV-1, inparticular associated with multiple sclerosis.

The techniques for the culture and detection of retroviral materialwhich were used in the work carried out by the Applicant on a multiplesclerosis (MS) associated agent are described in French PatentApplications 92 04322, 92 13447, 92 13443, 92 01529, 94 01530, 94 01531,94 01532 and in the publication by H. PERRON et al. (Res. Virol. 1989;140, 551-561) (the content of which is incorporated by reference intothe present description).

This retrovirus may be derived from a viral strain chosen from thestrains called, respectively, POL-2, deposited on 22.07.1992 at theECACC under the accession number V92072202, and MS7PG deposited on08.01.93 at the ECACC under the accession number V93010816, or producedby a cell line chosen from the lines called, respectively, PLI-2deposited on 22.07.1992 at the ECACC under the accession number92072201, and LM7PC deposited on 08.01.93 at the ECACC under theaccession number 93010817.

Among the polypeptides of the invention, the Applicant has determined apolypeptide (generically called pxMSRV-1 in the remainder of thedescription) capable of reacting specifically with at least onebiological fluid from a patient in whom the MSRV-1 viral sequences havebeen detected. According to the invention, this polypeptide possesses apeptide sequence which comprises at least one sequence chosen from SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and their equivalentsequences, said polypeptide being different from the polypeptides havingany one of the following sequences: SEQ ID NO: 20, SEQ ID NO: 21, SEQ IDNO: 25. Preferably, the sequence of the polypeptide pxMSRV-1 consists ofSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

Before disclosing the invention in greater detail, various terms used inthe description and the claims are defined below.

“Polypeptide” designates a peptide, in the isolated state, exhibiting asuccession of a variable number of amino acids, such as an oligopeptide,a protein, a fusion protein, a fusion peptide, a synthetic peptide. Apolypeptide may be obtained by various techniques well known to personsskilled in the art, and in particular by chemical synthesis or bygenetic recombination techniques. The polypeptides according to theinvention may be obtained by methods of conventional synthesis, forexample with an automated peptide synthesizer, or by genetic engineeringtechniques comprising the insertion of a DNA sequence encoding saidpolypeptide into an expression vector such as a plasmid or a virus, andthe transformation of cells with this expression vector and culture ofthese cells.

A polypeptide of the invention advantageously contains at most 50 aminoacids, preferably at most 30 amino acids, or better still at most 20amino acids, or even at most 15 amino acids.

“Peptide sequence equivalent to a reference peptide sequence isunderstood to mean an amino acid sequence modified by insertion and/ordeletion and/or substitution and/or extension and/or shortening and/orchemical modification of one or more amino acids, as long as thesemodifications substantially preserve or even develop the immunoreactiveproperties of said reference peptide sequence. Advantageously, saidequivalent sequence exhibits, for at least a succession of 6 aminoacids, a percentage identity of at least 40%, preferably of at least50%, or better still of at least 60% or even 70%, with said referencesequence. This percentage identity is calculated according to thefollowing steps: a succession of 6 contiguous amino acids of thesequence analyzed is compared with a succession of 6 contiguous aminoacids of the reference sequence, the amino acids which are commonbetween the analyzed sequence and the reference sequence, located at thesame position, are determined and the percentage identity is deduced.

“Equivalent sequence” is thus understood to mean in particular thesequences in which one or more amino acids are substituted by one ormore other amino acids; the sequences in which one or more amino acidsof the L series are replaced by an amino acid of the D series, and viceversa; the sequences in which a modification of the side chains of theamino acids, such as an acetylation of the amine functions, acarboxylation of the thiol functions, an esterification of the carboxylfunctions, is introduced; a modification of the peptide bonds such as,for example, the carba, retro, inverse, retro-inverse, reduced andmethyleneoxy bonds.

The equivalence of a peptide sequence relative to a reference peptidesequence may be defined by its identity or its homology, expressed as apercentage, with said reference sequence. This percentage is determined,for a succession of a given number of contiguous amino acids, byaligning the two sequences, moving one relative to the other, andcomparing the amino acids in the two sequences. The percentage identityis determined from the number of amino acids which are identical to theamino acids of the reference sequence, at the same position. Thepercentage homology is determined from the number of amino acids whichare equivalent to amino acids of the reference sequence, at the sameposition. Using the BLAST program (BLAST p matrix Blosum62) publiclyavailable (on the Internet, at the National Center for BiotechnologyInformation (NCBI), Bethesda, USA), persons skilled in the art are in aposition to know if the sequence which they have selected exhibits thepercentage homology required according to the invention, compared withthe reference sequence. In accordance with the BLAST program, two aminoacids are equivalent if they possess similar physicochemical properties,such as hydrophilicity, isoelectric point.

Viral sequence of the MSRV-1 virus is understood to mean in particularall the nucleotide sequences described in French Patent Applications 9204322, 92 13447, 92 13443, 92 01529, 94 01530, 94 01531, 94 01532, 9509643, in the name of the Applicant.

Binding of antibodies is understood to mean the separation, isolation,detection and/or quantification of these antibodies, the enrichment ofan antibody fraction, according to a specific or aspecific bindingmethod, qualitatively and/or quantitatively.

“Polynucleotide” is understood to mean either a DNA sequence or an RNAsequence or a cDNA sequence resulting from the reverse transcription ofan RNA sequence, of natural or synthetic origin, with or withoutmodified bases.

The invention additionally relates to:

a reagent for the detection of multiple sclerosis in a patient and/orthe monitoring of a patient suffering from multiple sclerosis,comprising at least one, or consisting of a, polypeptide capable ofreacting specifically with the antibodies of patients suffering frommultiple sclerosis (MS) and whose peptide sequence comprises at leastone sequence chosen from SEQ ID NO: 1 to SEQ ID NO: 19, and theirequivalent sequences, said polypeptide being optionally labeled; as wellas a kit comprising this reagent, the latter being supported on asupport which is immunologically compatible with said reagent;

a reagent for the detection of an MSRV-1 virus infection comprising atleast one, or consisting of a, polypeptide pxMSRV-1 capable of reactingwith at least one biological fluid from a patient in whom MSRV-1 viralsequences have been detected and whose peptide sequence comprises atleast one, or consists of a, sequence chosen from SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and their equivalent sequences, saidpolypeptide being optionally labeled; as well as a kit comprising thisabovementioned reagent optionally supported on a support which isimmunologically compatible with said reagent;

Advantageously, a reagent of the invention comprises at least twodifferent polypeptides as defined above and in particular threepolypeptides as defined above. In a particularly preferred form, areagent comprises the three polypeptides whose peptide sequence eachconsists of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 16,respectively.

a method of binding, in a biological sample, antibodies which arecharacteristic of and/or specific to multiple sclerosis, consisting inbringing said sample into contact with a reagent of the invention, andafter having optionally detected the presence of an immune complex, inseparating the latter;

a method of binding, in a biological sample, antibodies directed againstthe MSRV-1 virus, comprising the steps consisting is bringing saidsample into contact with a reagent of the invention comprising at leastone polypeptide pxMSRV-1, and after having optionally detected thepresence of an immune complex, in separating the latter;

according to a preferred embodiment of any of the methods of binding ofthe invention, the sample is chosen from serum, cerebrospinal fluid andurine;

an immunotherapeutically active composition, in particular a vaccinepreparation, comprising at least one polypeptide capable of reactingspecifically with the antibodies of patients suffering from multiplesclerosis (MS) and whose peptide sequence comprises at least onesequence chosen from SEQ ID NO: 1 to SEQ ID NO: 19, and their equivalentsequences, and optionally a support for said polypeptide and/or apharmaceutically acceptable excipient;

the use of at least one polypeptide capable of reacting specificallywith the antibodies of patients suffering from multiple sclerosis (MS)and whose peptide sequence comprises at least one sequence chosen fromSEQ ID NO: 1 to SEQ ID NO: 19, and their equivalent sequences, forbinding, in a biological sample, antibodies which are characteristic ofand/or specific to multiple sclerosis, and the use of at least onepolypeptide pxMSRV-1 of the invention for binding, in a biologicalsample, antibodies directed against the MSRV-1 virus; and

a polynucleotide whose nucleotide sequence comprises a sequence encodinga polypeptide of the invention.

BRIEF DESCRIPTION OF THE FIGURES

The invention disclosed above is now illustrated in the followingexamples 1 to 6 in support of FIGS. 1 to 7B according to which:

FIG. 1 represents the IgG response of the polypeptide SEQ ID NO: 3 withrespect to human sera, on a histogram grouping together the resultsexpressed in optical density values (×1000) and presented in Table 2appended to the description; the polypeptide is tested in ELISA withrespect to sera diluted 1/100; the MS− sera correspond to sera fromhealthy individuals; the MS+ sera correspond to sera from MS patientswho have never been treated and who are all at the remission stage.

FIG. 2 represents the IgG response of the polypeptide SEQ ID NO: 4 withrespect to human sera, on a histogram grouping together the resultsexpressed in optical density values (×1000) and presented in Table 3appended to the description; the polypeptide is tested in ELISA withrespect to sera diluted 1/100; the MS− sera correspond to sera fromhealthy individuals; the MS+ sera correspond to sera from MS patientswho have never been treated and who are all at the remission stage.

FIG. 3 represents the nucleotide sequence and its translation into aminoacids of the clone LB19; the succession of amino acids common with SEQID NO: 1 and SEQ ID NO: 3 is underlined;

FIG. 4 represents the partial nucleotide sequence and its translationinto amino acids of the clone GM3; the succession of amino acids commonwith SEQ ID NO: 2 and SEQ ID NO: 4 is underlined;

FIG. 5 represents the IgM response of the polypeptide SEQ ID NO: 8 withrespect to human sera, on a histogram grouping together the resultsexpressed in optical density values (×1000) and presented in Table 4appended to the description; the polypeptide is tested in ELISA withrespect to sera diluted 1/100; the MS− sera correspond to sera fromhealthy individuals; the MS+ sera correspond to sera from MS patientswho nave never been treated and who are all at the remission stage.

FIG. 6 represents the IgM response of the CSFs with respect to phagesexpressing the sequences SEQ ID NO: 14 (EPM), SEQ ID NO: 13 (FCP), SEQID NO: 16 (SRG), SEQ ID NO: 17 (QSP) , with respect to the wild-typephage (without an expressed sequence) and another nonspecific sequence(RTG); this representation is given by a histogram grouping together theresults expressed as optical density values obtained for each CSFreduced by those obtained with PBS buffer controls in place of thephages; the immunoreactivity of the phage clones is tested in ELISA asdescribed in Example 2, with respect to CSFs diluted 1/10; CSF 12corresponds to a patient suffering from a neurological disease otherthan MS; CSF 4 and CSF 7 correspond to two patients suffering from MS.

FIGS. 7A and 7B represents the IgG response of the polypeptides whosepeptide sequence consists of, respectively, SEQ ID NO: 13, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 19 and SEQ ID NO: 15, withrespect to CSFs diluted 1/10; the results assembled in the appendedTable 5 are expressed by the difference in the optical density valuesobtained for the polypeptide tested and the values obtained for the HIVpolypeptide used as control; ND corresponds to patients suffering from aneurological disease other than MS, and MS* corresponds to patientssuffering from MS.

EXAMPLE 1 Selection of Hexapeptides Capable of Reacting Specificallywith Sera from Patients Suffering from Multiple Sclerosis

In a first instance, a library for expression of hexapeptides wasconstructed in a filmentous phage according to the method described bySCOTT and SMITH (1990, Science, 249, 386-390). This library is producedby inserting a synthetic oligonucleotide into a gene encoding a phageenvelope protein (PIII protein) of which five copies are present at thesurface of the phage. This oligonucleotide consists of a sequence ofhaving a degenerate code [(NNK) 6] where NNK represents an equal mixtureof the codons corresponding to the 20 amino acids and the Amber stopcodon. This expression library makes it possible to obtain, at thesurface of the phage, five copies of a fused protein (PIII—hexapeptide).The site of insertion of the hexapeptide into the sequence of PIIIprotein corresponds to the sequence: NH₂ Ala Asp Gly Ala [hexapep] GlyAla Ala Gly Ala Glu Thr Val Glu COOH (SEQ ID NO: 29).

In a second instance, the bottom of a Petri dish 35 mm in diameter istreated with 1 ml of a streptavidin solution at the concentration of 10μg/ml in 0.1M NaHCO₃ and incubated overnight at 4° C. After removing thestreptavidin solution, a solution of 0.1M NaHCO₃, 0.1% of bovine serumalbumin (BSA), 0.1 μg/ml of streptavidin and 0.02% of NaN₃ is added inorder to saturate the nonspecific binding sites and the whole isincubated for 2 hours at room temperature. The Petri dish is washed 6times with TBS buffer (0.1M Tris buffer, pH 7.2)/0.5% Tween and 10 μg ofbiotinylated total human antiimmunoglobulins (Southern BiotechnologyAssociates Inc.) are then added and incubated for 4 hours at 4° C. Afteran additional 1 hour of incubation in the presence of 20 μl of 2 mMbiotin, the Petri dish is again washed 6 times with TBS/0.5% tween.

Moreover, 20 μl of serum (about 100 μg of total immunoglobulins)obtained from a patient suffering from multiple sclerosis at theremission stage (called patient No. 1) were preincubated for 8 hours at4° C., with stirring, with 50 μl (about 5×10¹² virions) of a solution ofwild-type phages, that is to say not containing an inserted peptidesequence. This makes it possible to eliminate any possible binding ofthe immunoglobulins to phage proteins other than the inserted peptidesequence. This mixture is added to the Petri dish treated as describedabove and incubated overnight at 4° C., with stirring. This makes itpossible to obtain, in a known manner, Petri dishes at the bottom ofwhich said antibodies are immobilized by means of streptavidin andbiotinylated human total antiimmunoglobulins.

After 10 new washes in TBS/0.5% tween buffer, a sample of the expressionlibrary containing about 10¹² virions is then incubated for four hoursat 4° C. in the presence of said antibodies bound to the bottom of thePetri dish. After several washes with TBS, the phages which remainedbound to the antibodies contained in the serum of patient No.1 areeluted with 400 μl of a 0.1N HCl solution, pH 2.2, containing 0.1% BSA,and then neutralized with 75 μl of a 1M Tris-HCl solution, pH 9.1.

After concentrating to 100 μl, the suspension of eluted phages issubjected to an amplification step by infecting a suspension at 5×10⁹bacteria/ml of a strain of E. coli (K91Kan) . The infected bacteria (seeSambrook et al., 1989. Molecular Cloning : A Laboratory Manual, 2ndEdition, Cold Spring Harbor Laboratory, Cold Spring Harbor) areincubated for 45 minutes at 37° C. in 20 ml of NZY medium (tryptone 10g/l, yeast extract 5 g/l, NaCl 5 g/l, pH adjusted to 7) containing 0.2μg/ml of tetracycline. The concentration of tetracycline in the mediumis then raised to 20 mg/ml. Given that the infectious phages carry atetracycline resistance gene, only the bacteria which have been infectedby the phage are amplified. The culture of the bacteria is continuedovernight at 37° C. After centrifugation of the culture in order toremove the bacterial cells, 3 ml of polyethylene glycol (PEG) 16.3%-NaCl3.3M are added to the supernatant in order to precipitate the phagespresent. After overnight incubation at 4° C. and a centrifugation, thephage pellet is taken up in 1 ml of TBS (0.1M Tris buffer, pH 7.2) andreprecipitated with 150 μl of PEG/NaCl. A centrifugation makes itpossible to obtain a phage pellet which is resuspended in 200 μl of TBS.The phage concentration after this amplification is about 2×10¹³virions/ml.

The 2nd selection or “biopanning 2” is carried out according to theprotocol described above using 20 μl of serum from patient No.2 andabout 10¹² virions derived from the Preceding amplification step.

Likewise, the 3rd selection will be carried out using, respectively, 20μl of serum from patient No.3 and 10¹² virions derived from theamplification of the 2nd selection.

The 4th selection will use 20 μl of serum from patient No.4 and 10¹²virions derived from the amplification of the 3rd selection.

The 5th selection is carried out differently: 20 μl of a pool of 5 seracorresponding to patient Nos. 5, 6, 7, 8, 9, 10 are preincubated with 50μl of wild-type phage for 8 hours at 4° C. before being deposited in thePetri dish and incubated overnight at 4° C., with stirring, so as to becaptured by the biotinylated human total antiimmunoglobulins. Moreover,100 μl of the amplified phages after the 4th selection (about 10¹²virions) were preincubated overnight at 4° C., with stirring, with 100μl of a pool of 5 sera from healthy individuals (that is to say about1.2 mg of total immunoglobulins) . After 10 washes of the Petri dishwith TBS/0.5% Tween, the preceding mixture: phages derived from the 4thselection and sera from healthy individuals is brought into contact, inthe Petri dish, with the total immunoglobulins of the pool of patients.The immunoglobulins bound in the Petri dish and nonspecific to thepatient will thus be in competition with a large excess of the sameimmunoglobulins present in the mixture to interact with the phages. Onlythe selection of the phages interacting with the immunoglobulinsspecific to the patient will be favored. After 10 new washes of thePetri dish, the phages bound are eluted and the eluate neutralized asdescribed for the 1st selection.

10 μl of the 10⁻⁸ and 10⁻⁹ dilutions of the phages eluted above are eachused to infect 10 μl of a suspension containing 5×10⁹ bacteria/ml of theK91Kan strain. After incubating for 10 min at room temperature, 1 ml ofNZY medium containing 0.2 μg of tetracycline are added for an additionalincubation of 45 minutes at 37° C., with stirring. The suspensions ofbacterial infected with the phages are then plated in Petri dishes (85mm in diameter), on solid NZY medium containing 40 μg/ml of tetracyclineand 100 μg/ml of kanamycin.

After incubating for 24 hours at 37° C., 10⁸ randomly chosen infectedbacteria colonies are inoculated individually into 1.7 ml ofNZY-tetracycline (20 μg/ml) medium. After culturing, with stirring, for16 to 24 hours at 37° C., the cells are removed by centrifugation. Thesupernatant (1 ml) is mixed with 150 μl of a PEG/NaCl solution andincubated for four hours at 4° C. After centrifugation, the phages areresuspended in 500 μl of TBS.

The phage preparations thus obtained contain about 5×10¹¹ phages permilliliter.

EXAMPLE 2 Immunological Analysis of Said Clones by the ELISA Technique

Each clone is tested simultaneously for its IgG and IgM immunoreactivitywith respect to a pool of 5 sera from healthy individuals and a pool ofsera from patients suffering from multiple sclerosis at the remissionstage. Each trial is carried out in triplicate.

In a first instance, 100 μl of anti-M13 phage antibodies diluted 1/500in 0.1M NaHCO₃, pH 8.6 (marketed by Prinn, Inc. Boulder, Colo. 80303)are bound overnight in the wells of Nunc maxisorb (trade name)microtiter plates. After three washes with TBS/0.05% Tween, the platesare passivated for 1 hour at 37° C. with 250 μl of TBS containing 10%goat serum and again washed three times with the same buffer. 100 μl ofvarious purified phage clones are then incubated for 2 hours at 37° C.in the wells. After three washes with TBS/0.05% Tween, 100 μl of a 1/50dilution of serum are added and incubated for 2 hours at 37° C. Afterfour washes in TBS/0.05% Tween, 100 μl of peroxidase-labeled anti-humanIgG conjugate diluted 1/10,000 (marketed by Jackson Immuno ResearchLaboratories Inc.) are added before an incubation for one hour at 37° C.The enzymatic reaction for revealing is carried out by adding 100 μl ofan H₂O₂/ortho-phenylenediamine (OPD) solution and incubating the samplefor 30 minutes at room temperature. The staining reaction is interruptedby the addition of 50 μl of 1.8N sulfuric acid. The optical density ismeasured on a BioMérieux plate reader at 492 nm. The IgM response isdetermined by addition of 100 μl of biotinylated anti-IgM conjugatediluted 1/4000 (Biomérieux monoclonal antibody) followed by theaddition, after the washes, of the streptavidin-peroxidase conjugatediluted 1/10,000. The revealing reaction is then carried out asdescribed above.

The results obtained in ELISA are expressed by subtracting, for eachclone, the mean of the values obtained with the pool of sera fromhealthy individuals from the mean of the values obtained with the poolof sera from patients. A positive response is thus obtained for 37 phageclones.

EXAMPLE 3 Determination of the Sequence of the Phage Clones Selected

The DNA of the phages corresponding to the 37 clones which gave apositive response in ELISA was prepared according to the methoddescribed by Sambrook et al., 1989. Molecular Cloning : A LaboratoryManual, 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor.

These clones were sequenced using a prime of 20 bases(5′-CCCTCATAGTTAGCGTAACG-3′) SEQ ID NO: 28. This primers iscomplementary to nucleotides 1717-1736 of the gene encoding the nativePIII protein of the phage. The sequencing was carried out on anautomated sequencer (Applied Biosystems, 373 A) using the “Prism readyreaction kit dye deoxyterminator cycle sequencing dye” according to thesupplier's protocol (Applied Biosystems).

The nucleic sequences obtained, when they are converted to amino acidsequences, indicate that the peptide sequences SEQ ID NO: 3 (Leu Gln GlnAla Val Phe) and SEQ ID NO: 4 (Ser Thr Gly Arg Pro Leu) are found 11times and 6 times, respectively, in the clones selected for theirpositive response. Furthermore, as shown in the following table, othersequences are represented in duplicate or in triplicate.

Moreover, the sequence of the clones for which a negative response isobserved does not possess any homology with these sequences.

The following Table 1 gives the IgG and IgM responses of the variousphage clones expressing the sequences SEQ ID NOs: 3 to 9.

The results are expressed by subtracting, for each clone, the mean ofthe values obtained with the pool of sera from healthy individuals fromthe mean of the values obtained with the pool of sera from patients.

TABLE 1 No. of Peptide seq. clones IgG rp IgM rp SEQ ID NO: 3 11  0.1350    LQQAVF 0.160 0.009 0.266 0    0.335 0.074 0.105 0.097 0.280 0.0560.267 0.061 0.137 0.043 0.113 0.026 0.383 0.069 0.310 0.067 SEQ ID NO: 46 0.264 0    STGRPL 0.182 0.007 0.160 0    0.131 0.026 0.110 0.054 0.2870.023 SEQ ID NO: 5 2 0.185 0.068 RLVLVP 0.366 0.012 SEQ ID NO: 6 3 0.0580.042 FLENGV 0.016 0.042 0.020 0.044 SEQ ID NO: 7 2 0.206 0.066 KGTSLS0.116 0.017 SEQ ID NO: 8 1 0.119 0.506 LAVRHD SEQ ID NO: 9 1 0.280 0.247TFDRRI

EXAMPLE 4 Chemical Synthesis of Two Pentadecapeptides and ELISA Testwith Positive Human Sera

Depending on the information obtained from the preceding examples, thehexapeptides SEQ ID NO: 3 and SEQ ID NO: 4 were synthesized andbiotinylated according to the method described in Patent Application No.EP 93420183 with the following modifications: the peptide still bound tothe resin is selectively deprotected at the N-terminal position. Afterovernight incubation with the biotin-NHS at 50% in DMF, the peptide isthen cleaved from the resin and treated as described in the patent citedabove.

These peptides were then tested in ELISA with respect to theirreactivity towards several sera from multiple sclerosis patients andfrom healthy individuals.

The wells of a Maxisorb (trade name, Nunc) microtiter plate aresaturated with 100 μl of a 50 mM NaHCO₃ solution, pH 9.6, containing 10μg/ml of streptavidin for 2 hours at 37° C. The plate is washed withTBS/0.05% Tween 20 buffer. The wells are then saturated with 250 μl ofwash buffer supplemented with 10% goat serum for 1 hour at 37° C. andthen washed as described above.

The sera to be analyzed are diluted 1/100 in saturation buffer,supplemented with 0.05% Tween 20 and incubated for 2 hours at 37° C.After washing, a solution of peroxidase-labeled anti-humanimmunoglobulin G goat antibody conjugate (marketed by Jackson ImmunoResearch Laboratories Inc.) is added at the dilution of 1/10,000 and theplates are incubated for 1 hour at 37° C. After washes, theortho-phenylenediamine (OPD) substrate is added and the reaction isstopped after 10 minutes by adding 50 μl of H₂SO₄. The optical densityis read at 492 nm.

The IgM response is determined by addition of a 1/4000 solution of mousemonoclonal antibody anti-alkaline phosphatase-labeled human Mimmunoglobulin conjugate (bioMérieux Ac 5A10D5). After incubation at 37°C. for one hour the plate is again washed and the p-nitrophenylphosphate substrate at 1 mg/ml in an M solution of diethanolamine, pH9.8, containing 1 mM MgCl₂, is added. The reaction is stopped after 30minutes by the addition of 50 μl of 3N sodium hydroxide solution. Theoptical density is read at 405 nm.

In a test carried out with 1/100 dilutions of 11 English sera from MSpatients at the remission stage and 4 negative English sera, thepeptides SEQ ID NOs: 3 and 4 are recognized by 4 sera out of 11 whichgive a positive IgG signal compared with the negative serum which gavethe highest signal. If the mean of the values obtained with the negativesera is calculated and 3 times the standard deviation is added in orderto determine a cut-off, the 2 peptides are still recognizedsignificantly by 3 sera out of 11 (FIGS. 1 and 2).

The sequence SEQ ID NO: 3 has 4 consecutive and identical amino acids inthe MSRV-1 region encoded by the LB19 clone (represented in FIG. 3).Likewise, the sequence SEQ ID NO: 4 has 4 consecutive and identicalamino acids in the MSRV-1 region encoded by the GM3 clone (representedin FIG. 4).

The unit SEQ ID NO: 8 was found only once after sequencing, however, itgave a positive IgM response and exhibits sequence homology with HTLV1gag. This unit was therefore also synthesized and biotinylated. The IgMresponse tested with respect to the same panel of sera described aboveshows that 7 sera out of 11 recognize this peptide (FIG. 5).

EXAMPLE 5 Selection of Pentadecapeptides Capable of ReactingSpecifically with Sera from Patients Suffering from Multiple Sclerosis

A library of pentadecapeptides carried on 300 copies of the phageprotein VIII was constructed on the same principle as the hexapeptidelibrary using the vector f88-4 according to the work by Greewood et al.(J. Biol. Mol. 1991). When f88-4 is propagated in the presence of IPTG,the foreign pentapeptide is expressed on the whole surface of thevirion.

The same selection protocol as that described in Example 1 was used toscreen this pentadecapeptide library with new remission sera.

After 5 biopannings, 72 phage clones were immunoanalyzed: 39 cloneswhich gave a positive signal with respect to a pool of positive seracompared with a pool of negative sera were sequenced:

17/39 carry the sequence SEQ ID NO: 10: Asn Ala Cys Tyr Val Asp Leu PheLeu Gly Ala Ser Val Cys Pro, these clones give an average positive IgGresponse of 0.069 with a 1/200 serum dilution

12/39 clones carry the sequence SEQ ID NO: 11: Ser Ser Ala Lys Ser HisCys Tyr Ala Phe Cys Ser Gly Leu Pro, with an average positive responseof 0.248.

It should be noted that these 2 units both carry the amino acidsCys-Tyr. Furthermore, according to the BLAST program, SEQ ID NO: 11shares 62% identity and 87% homology with the foot-and-mouth diseasevirus SAT3 protein.

Moreover, 2/39 clones carrying the sequence SEQ ID NO: 18 also have anaverage positive response of 0.358.

EXAMPLE 6 Selection of Pentadecapeptides Capable of ReactingSpecifically with Cerebrospinal Fluids from Patients Suffering fromMultiple Sclerosis

The same pentadecapeptide library was targeted using the cerebrospinalfluids (CSF) from untreated patients suffering from multiple sclerosis,at the remission or chronic progressive phase. These CSFs were selectedas a function of their oligoclonal profile by isoelectric focusing andof their IgG index, these two parameters showing an intrathecalsynthesis of IgG.

In a protocol A, 4 biopannings were carried out with the CSFs from 4different patients according. to the principle described in Example 1.41 phage clones were then isolated and their DNA sequenced by automatedsequencing using a primer of 20 bases (5′-TGAAGAGAGTCAAAAGCAGC-3′)specific for protein VIII.

The units obtained are the following:

MPVSRLCIELDWCPP 4/41 SEQ ID NO: 12 FCPPILPYSAWCPVP 4/41 SEQ ID NO: 13EPMTPHQWITLYRSY 15/41  SEQ ID NO: 14 DTPYPWGWLLDEGYD 9/41 SEQ ID NO: 15RGTQEWTELWVSFRA 2/41 SEQ ID NO: 19

In parallel, in a protocol B, 4 successive biopannings were carried outusing the same CSF (CSF 4 used for the first biopanning of protocol A)at increasing dilutions: 1, 1/10, 1/20 and 1/100.

32 phage clones derived from protocol B were also isolated andsequenced. The results show that the 2 units obtained with protocol Aare also found with protocol B using a 1/100 dilution of CSF.

EPMTPHQWITLYRSY 21/32  SEQ ID NO: 14 FCPPILPYSAWCPVP 2/32 SEQ ID NO: 13SRGSHEWAVLFRFYY 3/32 SEQ ID NO: 16 RGTQEWTELWVSFRA 2/32 SEQ ID NO: 19QSPLEDRILRFLSPP 2/32 SEQ ID NO: 17

The phage clones carrying the sequences SEQ ID Nos: 13, 14, 16 and 17 aswell as the nonrecombinant phage (that is to say not containing aninserted pentadecapeptide), were tested in ELISA with respect to a CSFfrom a neurological patient, CSF 4 (used in protocol B and the 1stbiopanning of protocol A) and CSF 7 used for the 2nd biopanning ofprotocol A.

FIG. 6 shows that the sequence SEQ ID NO: 13 is recognized specificallyby CSFs 7 and 4, whereas SEQ ID NO: 14 is recognized only by CSF 7.However, the predominant unit is not recognized by any of these twoCSFs.

The sequence SEQ ID NO: 13 appears to be advantageous since, accordingto BLAST program, it shares 58% identity and 75% homology with thesequence p30/p10/5′v-fsm of the coding region of the feline sarcomavirus [NCBI reference gi/554646].

In a similar experiment, the pentadecapeptide library was screened inthe same order of use with the sera obtained from patients whose CSFswere used above.

In a manner identical to Example 5, the sequences SEQ ID NO: 10 (18/36),SEQ ID NO: 11 (2/36) and SEQ ID NO: 18 (12/36) are again predominantlyfound. None of the other sequences selected corresponds to thoseselected by the CSFs and vice versa.

The difference in relative proportions of the clones compared withExample 5 may be explained by the fact that the sera used here areobtained from patients predominantly at a chronic stage of the diseasewhereas in Example 5 all the patients are at a remission stage.

Table 5, illustrated in FIGS. 7A and 7B, collates the results on thespecific recognition of CSF from patients suffering from MS for 6polypeptides of the invention, SEQ ID NO: 13, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 19 and SEQ ID NO: 15.

It is evident from Table 5 that the combination of the mostimmunoreactive polypeptides, namely SEQ ID NO: 12, SEQ ID NO: 13 and SEQID NO: 16, makes it possible to detect specific antibodies in 13 samplesof CSF from patients suffering from MS out of 15.

TABLE 2 IGG RESPONSE TO THE BIOTINYLATED PEPTIDE L4F SERUM OD 1 OD 2 OD3 MEAN BASELINE MS − Control 1 15 18 17 17 815 No. 12 550 640 726 639No. 13 366 369 407 377 No. 14 367 285 270 307 No. 15 439 496 422 452MS + 33/6  535 599 626 587 33/8  990 941 1271 1067 X 33/18 601 339 246395 QS 2 294 339 293 309 QS 3 618 784 779 727 QS 4 1161 1293 1583 1346 XQS 6 306 263 407 325 QS 7 1307 1298 1461 1355 X QS 8 664 635 545 615  QS11 429 169 108 235  QS 12 405 472 316 398

TABLE 3 IGG RESPONSE TO THE BIOTINYLATED PEPTIDE S4L SERUM OD 1 OD 2 OD3 MEAN BASELINE MS − Control 1 16 17 37 23 674 No. 12 567 518 469 518No. 13 286 278 268 271 No. 14 322 401 354 359 No. 15 423 432 470 442MS + 33/6  465 496 525 496 33/8  849 820 753 807 X 33/18 258 243 299 267QS 2 267 296 267 277 QS 3 668 618 585 624 QS 4 1096 1120 862 1026 X QS 6204 212 201 206 QS 7 1379 1437 1323 1380 X QS 8 523 504 630 552  QS 11272 273 261 269  QS 12 271 227 212 237

TABLE 4 IGM RESPONSE TO THE BIOTINYLATED PEPTIDE L4D SERUM OD 1 OD 2 OD3 MEAN MEAN LINE MS − Control 1 213 170 136 173 173 No. 12 222 175 163187 14 No. 13 145 154 196 165 −6 No. 14 320 165 143 209 36 No. 15 254205 173 211 38 MS + 33/6  516 529 481 509 336 X 33/8  609 512 568 563390 X 33/18 277 262 255 265 92 X QS 2 281 280 231 264 91 X QS 3 191 169164 175 2 QS 4 374 361 346 360 187 X QS 6 156 147 151 151 −22 QS 7 311290 164 265 82 X QS 8 305 221 265 264 91 X  QS 11 164 160 139 154 −19 QS 12 123 57 148 109 −64

TABLE 5 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID Patients NO: 13 NO: 12NO: 14 NO: 16 NO: 19 NO: 15 1 + + + + − − 2 + + + + − − 3 + − + + − −5 + + + + + − 6 − + − + + − 7 + + + + + − 8 + + − + + − 10 + + + + + −16 − + − − − − 17 − − − − − − 18 + + + + + − 19 − − − − − − 20 + − − − −− 21 + + + + + − 22 − − − + + − 15 10+ 10+ 8+ 11+ 8+ 0+

For each polypeptide tested, identified in Table 5 by its peptidesequence, the CSFs from patients 1-22 suffering from Ms are determinedas positive (+) if the OD value at 492 nm for a 1/10 dilution of the CSFis greater than the mean values obtained for the CSFs from the NDpatients+3 times the standard deviation.

34 1 4 PRT MSRV 1 Gln Gln Ala Val 1 2 4 PRT MSRV 2 Thr Gly Arg Pro 1 3 6PRT MSRV 3 Leu Gln Gln Ala Val Phe 1 5 4 6 PRT MSRV 4 Ser Thr Gly ArgPro Leu 1 5 5 6 PRT MSRV 5 Arg Leu Val Leu Val Pro 1 5 6 6 PRT MSRV 6Phe Leu Glu Asn Gly Val 1 5 7 6 PRT MSRV 7 Lys Gly Thr Ser Leu Ser 1 5 86 PRT MSRV 8 Leu Ala Val Arg His Asp 1 5 9 6 PRT MSRV 9 Thr Phe Asp ArgArg Ile 1 5 10 15 PRT MSRV 10 Asn Ala Cys Tyr Val Asp Leu Phe Leu GlyAla Ser Val Cys Pro 1 5 10 15 11 15 PRT MSRV 11 Ser Ser Ala Lys Ser HisCys Tyr Ala Phe Cys Ser Gly Leu Pro 1 5 10 15 12 15 PRT MSRV 12 Met ProVal Ser Arg Leu Cys Ile Glu Leu Asp Trp Cys Pro Pro 1 5 10 15 13 15 PRTMSRV 13 Phe Cys Pro Pro Ile Leu Pro Tyr Ser Ala Trp Cys Pro Val Pro 1 510 15 14 15 PRT MSRV 14 Glu Pro Met Thr Pro His Gln Trp Ile Thr Leu TyrArg Ser Tyr 1 5 10 15 15 15 PRT MSRV 15 Asp Thr Pro Tyr Pro Trp Gly TrpLeu Leu Asp Glu Gly Tyr Asp 1 5 10 15 16 15 PRT MSRV 16 Ser Arg Gly SerHis Glu Trp Ala Val Leu Phe Arg Phe Tyr Tyr 1 5 10 15 17 15 PRT MSRV 17Gln Ser Pro Leu Glu Asp Arg Ile Leu Arg Phe Leu Ser Pro Pro 1 5 10 15 1815 PRT MSRV 18 His Cys Arg Lys Val Thr Gly Ser Asp Tyr Leu Leu Cys GlyLeu 1 5 10 15 19 15 PRT MSRV 19 Arg Gly Thr Gln Glu Trp Thr Glu Leu TrpVal Ser Phe Arg Ala 1 5 10 15 20 565 PRT MSRV 20 Met Lys Met Arg Pro ArgTyr Ser Val Ile Ala Ser Ala Val Ser Leu 1 5 10 15 Gly Phe Val Leu SerLys Ser Val Met Ala Leu Gly Gln Pro Asp Thr 20 25 30 Gly Ser Leu Asn ArgGlu Leu Glu Gln Arg Gln Ile Gln Ser Glu Ala 35 40 45 Lys Pro Ser Gly GluLeu Phe Asn Gln Thr Ala Asn Ser Pro Tyr Thr 50 55 60 Ala Gln Tyr Lys GlnGly Leu Lys Phe Pro Leu Thr Gln Val Gln Ile 65 70 75 80 Leu Asp Arg AsnAsn Gln Glu Val Val Thr Asp Glu Leu Ala His Ile 85 90 95 Leu Lys Asn TyrVal Gly Lys Glu Val Ser Leu Ser Asp Leu Ser Asn 100 105 110 Leu Ala AsnGlu Ile Ser Glu Phe Tyr Arg His Asn Asn Tyr Leu Val 115 120 125 Ala LysAla Ile Leu Pro Pro Gln Glu Ile Glu Gln Gly Thr Val Lys 130 135 140 IleLeu Leu Leu Lys Gly Asn Val Gly Glu Ile Arg Leu Gln Asn His 145 150 155160 Ser Ala Leu Ser Asn Lys Phe Val Ser Arg Leu Ser Asn Thr Thr Val 165170 175 Asn Thr Ser Glu Phe Ile Leu Lys Asp Glu Leu Glu Lys Phe Ala Leu180 185 190 Thr Ile Asn Asp Val Pro Gly Val Asn Ala Gly Leu Gln Leu SerAla 195 200 205 Gly Lys Lys Val Gly Glu Ala Asn Leu Leu Ile Lys Ile AsnAsp Ala 210 215 220 Lys Arg Phe Ser Ser Tyr Val Ser Val Asp Asn Gln GlyAsn Lys Tyr 225 230 235 240 Thr Gly Arg Tyr Arg Leu Ala Ala Gly Thr LysVal Ser Asn Leu Asn 245 250 255 Gly Trp Gly Asp Glu Leu Lys Leu Asp LeuMet Ser Ser Asn Gln Ala 260 265 270 Asn Leu Lys Asn Ala Arg Ile Asp TyrSer Ser Leu Ile Asp Gly Tyr 275 280 285 Ser Thr Arg Phe Gly Val Thr AlaAsn Tyr Leu Asp Tyr Lys Leu Gly 290 295 300 Gly Asn Phe Lys Ser Leu GlnSer Gln Gly His Ser His Thr Leu Gly 305 310 315 320 Ala Tyr Leu Leu HisPro Thr Ile Arg Thr Pro Asn Phe Arg Leu Ser 325 330 335 Thr Lys Val SerPhe Asn His Gln Asn Leu Thr Asp Lys Gln Gln Ala 340 345 350 Val Tyr ValLys Gln Lys Arg Lys Ile Asn Ser Leu Thr Ala Gly Ile 355 360 365 Asp GlySer Trp Asn Leu Ile Lys Asp Gly Thr Thr Tyr Phe Ser Leu 370 375 380 SerThr Leu Phe Gly Asn Leu Ala Asn Gln Thr Ser Glu Lys Lys His 385 390 395400 Asn Ala Val Glu Asn Phe Gln Pro Lys Ser His Phe Thr Val Tyr Asn 405410 415 Tyr Arg Leu Ser His Glu Gln Ile Leu Pro Lys Ser Phe Ala Phe Asn420 425 430 Ile Gly Ile Asn Gly Gln Phe Ala Asp Lys Thr Leu Glu Ser SerGln 435 440 445 Lys Met Leu Leu Gly Gly Leu Ser Gly Val Arg Gly His GlnAla Gly 450 455 460 Ala Ala Ser Val Asp Glu Gly His Leu Ile Gln Thr GluPhe Lys His 465 470 475 480 Tyr Leu Pro Val Phe Ser Gln Ser Val Leu ValSer Ser Leu Phe Tyr 485 490 495 Asp Tyr Gly Leu Gly Lys Tyr Tyr Lys AsnSer Gln Phe Leu Glu Gln 500 505 510 Gly Val Lys Asn Ser Val Lys Leu GlnSer Val Gly Ala Gly Leu Ser 515 520 525 Leu Ser Asp Ala Gly Ser Tyr AlaIle Asn Val Ser Val Ala Lys Pro 530 535 540 Leu Asp Asn Asn Ile Asn AsnAla Asp Lys His Gln Phe Trp Leu Ser 545 550 555 560 Met Ile Lys Thr Phe565 21 359 PRT MSRV 21 Met Ala Lys Leu Leu Asp Ile Val Lys Pro Gly ValVal Thr Gly Glu 1 5 10 15 Asp Val Gln Lys Val Phe Ala Tyr Ala Lys GluHis Asn Phe Ala Ile 20 25 30 Pro Ala Val Asn Cys Val Gly Ser Asp Ser ValAsn Ala Val Leu Glu 35 40 45 Thr Ala Ala Arg Val Lys Ala Pro Val Ile IleGln Phe Ser Asn Gly 50 55 60 Gly Ala Ala Phe Tyr Ala Gly Lys Gly Ile LysPro Thr Ser Gly Thr 65 70 75 80 Arg Pro Asp Val Leu Gly Ala Ile Ala GlyAla Lys Gln Val His Thr 85 90 95 Leu Ala Lys Glu Tyr Gly Val Pro Val IleLeu His Thr Asp His Ala 100 105 110 Ala Lys Lys Leu Leu Pro Trp Ile AspGly Leu Leu Asp Ala Gly Glu 115 120 125 Lys His Phe Ala Glu Thr Gly ArgPro Leu Phe Ser Ser His Met Ile 130 135 140 Asp Leu Ser Glu Glu Ser MetGlu Glu Asn Met Ala Ile Cys Arg Glu 145 150 155 160 Tyr Leu Ala Arg MetAsp Lys Met Gly Met Thr Leu Glu Ile Glu Ile 165 170 175 Gly Ile Thr GlyGly Glu Glu Asp Gly Val Asp Asn Ser Asp Val Asp 180 185 190 Glu Ser ArgLeu Tyr Thr Gln Pro Ser Asp Val Leu Tyr Val Tyr Asp 195 200 205 Gln LeuHis Pro Val Ser Pro Asn Phe Thr Val Ala Ala Ala Phe Gly 210 215 220 AsnVal His Gly Val Tyr Lys Pro Gly Asn Val Lys Leu Lys Pro Ser 225 230 235240 Ile Leu Gly Glu Ser Gln Glu Phe Val Ser Lys Glu Arg Asn Leu Pro 245250 255 Ala Lys Pro Ile Asn Phe Val Phe His Gly Gly Ser Gly Ser Ser Arg260 265 270 Glu Glu Ile Arg Glu Ala Ile Gly Tyr Gly Ala Ile Lys Met AsnIle 275 280 285 Asp Thr Asp Thr Gln Trp Ala Ser Trp Asn Gly Ile Leu AsnPhe Tyr 290 295 300 Lys Ala Asn Glu Ala Tyr Leu Gln Gly Gln Leu Gly AsnPro Glu Gly 305 310 315 320 Pro Asp Ala Pro Asn Lys Lys Tyr Tyr Asp ProArg Val Trp Leu Arg 325 330 335 Lys Met Glu Glu Ser Met Ser Lys Arg LeuGlu Gln Ser Phe Glu Asp 340 345 350 Leu Asn Cys Val Asp Val Leu 355 22304 PRT MSRV 22 Met Thr Lys Met Lys Gln Met Leu Ile Cys Ile Leu Cys GlyHis Leu 1 5 10 15 Cys Ile Thr Trp Ile Gln Met Met Cys Gly Ile Arg GlnGln Val Gly 20 25 30 Ser Ile Lys Leu Ala Phe Thr Thr Tyr Met Glu His LeuAsn Thr Ile 35 40 45 Met Cys Tyr Leu Leu Met Met Gln Arg Asp Ile Val LeuLeu Glu Asn 50 55 60 Gly Lys Leu Lys Leu Ile Arg Lys Leu Cys Leu Leu LeuSer Leu Ala 65 70 75 80 Pro His His Gln Gly His Gln Glu Asp Lys Gln ThrGln Thr Pro Pro 85 90 95 Pro Arg Pro Pro Pro Pro Pro Gln Pro Pro Leu ThrPro Arg Pro Asp 100 105 110 Ala Asn Pro Ser Ile Asn Ser His Asn Lys ProLys Pro Asn Glu Glu 115 120 125 Gly Thr Asp Gly Asp His Gln Ala Glu GlnGly Asp Arg Lys Arg Thr 130 135 140 Lys Gly Asp Pro Asp Pro Asp Pro GlyArg Gly Pro Val Leu Lys Pro 145 150 155 160 Thr Leu Pro Pro Pro Pro ProPro Pro Pro Thr Gly Pro Gly Leu Arg 165 170 175 Arg Ser Thr Arg Leu ValLeu Val Pro Gly Gln Gly Pro Pro Pro Asp 180 185 190 Leu Pro Ala Pro ProVal Glu Gly Glu Val Glu Gly His Pro Gln Gly 195 200 205 Lys Asp Arg AspHis Pro Pro Pro Thr Pro Gln Asn Gly His Gly Lys 210 215 220 Glu Thr GlnGly Ala Glu Gly Gly Gly Asp Gln Gly Glu Gln Gly Ala 225 230 235 240 ValGly Gly Glu Ser Ser Asp Gly Glu Gly Asp His Ser Gln Pro Pro 245 250 255Leu Thr Pro Pro Asn Glu Ser Asp Gly Ser Leu Leu Asn Thr Val Ala 260 265270 Cys Leu Leu Ala Arg Trp Glu Ser Asn Phe Asp Gln Leu Val Gln Asn 275280 285 Ile Gln Gly Asp Leu Glu Gly Tyr Trp Arg Lys Leu Gly Thr Pro Gln290 295 300 23 425 PRT MSRV 23 Met Arg Gln Val Ala Tyr Arg Arg Arg ArgGlu Ser Ser Cys Ala Val 1 5 10 15 Leu Val His His Val Gly Arg Asp GlyAsp Gly Glu Gly Glu Ala Ala 20 25 30 Lys Lys Thr Cys Lys Lys Thr Gly ArgSer Val Ala Gly Ile Pro Gly 35 40 45 Glu Lys Leu Arg Arg Thr Val Val ThrThr Thr Pro Ala Arg Arg Leu 50 55 60 Ser Gly Arg His Thr Glu Gln Glu GlnAla Gly Met Arg Leu Cys Glu 65 70 75 80 Lys Gly Lys Lys Arg Ile Ile MetCys Arg Arg Glu Ser Leu Arg Thr 85 90 95 Leu Pro Trp Leu Phe Trp Val LeuLeu Ser Cys Pro Arg Leu Leu Glu 100 105 110 Tyr Ser Ser Ser Ser Phe ProPhe Ala Thr Ala Asp Ile Ala Glu Lys 115 120 125 Met Trp Ala Glu Asn TyrGlu Thr Thr Ser Pro Ala Pro Val Leu Val 130 135 140 Ala Glu Gly Glu GlnVal Thr Ile Pro Cys Thr Val Met Thr His Ser 145 150 155 160 Trp Pro MetVal Ser Ile Arg Ala Arg Phe Cys Arg Ser His Asp Gly 165 170 175 Ser AspGlu Leu Ile Leu Asp Ala Val Lys Gly His Arg Leu Met Asn 180 185 190 GlyLeu Gln Tyr Arg Leu Pro Tyr Ala Thr Trp Asn Phe Ser Gln Leu 195 200 205His Leu Gly Gln Ile Phe Ser Leu Thr Phe Asn Val Ser Met Asp Thr 210 215220 Ala Gly Met Tyr Glu Cys Val Leu Arg Asn Tyr Ser His Gly Leu Ile 225230 235 240 Met Gln Arg Phe Val Ile Leu Thr Gln Leu Glu Thr Leu Ser ArgPro 245 250 255 Asp Glu Pro Cys Cys Thr Pro Ala Leu Gly Arg Tyr Ser LeuGly Asp 260 265 270 Gln Ile Trp Ser Pro Thr Pro Trp Arg Leu Arg Asn HisAsp Cys Gly 275 280 285 Thr Tyr Arg Gly Phe Gln Arg Asn Tyr Phe Tyr IleGly Arg Ala Asp 290 295 300 Ala Glu Asp Cys Trp Lys Pro Ala Cys Pro AspGlu Glu Pro Asp Arg 305 310 315 320 Cys Trp Thr Val Ile Gln Arg Tyr ArgLeu Pro Gly Asp Cys Tyr Arg 325 330 335 Ser Gln Pro His Pro Pro Lys PheLeu Pro Val Thr Pro Ala Pro Pro 340 345 350 Ala Asp Ile Asp Thr Gly MetSer Pro Trp Ala Thr Arg Gly Ile Ala 355 360 365 Ala Phe Leu Gly Phe TrpSer Ile Phe Thr Val Cys Phe Leu Cys Tyr 370 375 380 Leu Cys Tyr Leu GlnCys Cys Gly Arg Trp Cys Pro Thr Pro Gly Arg 385 390 395 400 Gly Arg ArgGly Gly Glu Gly Tyr Arg Arg Leu Pro Thr Tyr Asp Ser 405 410 415 Tyr ProGly Val Arg Lys Met Lys Arg 420 425 24 96 PRT MSRV 24 Tyr Leu Arg HisThr Glu Glu Tyr Glu Val Glu Leu Ile Leu Arg Leu 1 5 10 15 Cys Lys ValPro Leu Asn Pro Asp Val Leu Ala His Leu Asn Val Met 20 25 30 Asp Lys AsnIle Leu Glu Asp Trp Gln Leu Ser Phe Val Pro Pro Pro 35 40 45 Pro Gln GlyIle Glu Asp Ala Tyr Arg Tyr Ile Met Ser Gln Ala Thr 50 55 60 Met Cys ProThr Asp Val Pro Asn Thr Glu Arg Glu Asp Pro Tyr Lys 65 70 75 80 Gln TyrThr Phe Trp Thr Ile Asp Leu Gln Glu Arg Phe Ser Asn Glu 85 90 95 25 64PRT MSRV 25 Gly Arg Asp Gly Tyr Ile Val Asp Ser Lys Asn Cys Val Tyr HisCys 1 5 10 15 Tyr Pro Pro Cys Asp Gly Leu Cys Lys Lys Asn Gly Ala LysSer Gly 20 25 30 Ser Cys Gly Phe Leu Val Pro Ser Gly Leu Ala Cys Trp CysAsn Asp 35 40 45 Leu Pro Glu Asn Val Pro Ile Lys Asp Pro Ser Asp Asp CysHis Lys 50 55 60 26 9 PRT MSRV 26 Lys Arg Asp Ser Ile Ser Pro Tyr Ser 15 27 9 PRT MSRV 27 Arg Arg Asp Thr Ile Ser Pro Tyr Ser 1 5 28 20 DNAUnknown Organism Description of Unknown Organism Primer 28 ccctcatagttagcgtaacg 20 29 19 PRT Unknown Organism CHAIN (5)..(10) Hexapeptidefrom Phage Library 29 Ala Asp Gly Ala Xaa Xaa Xaa Xaa Xaa Xaa Gly AlaAla Gly Ala Glu 1 5 10 15 Thr Val Glu 30 498 DNA MSRV 30 ctccttccccaactaataag gacccccctt tcaacccaaa cagtccaaaa ggacatagac 60 aaaggagtaaacaatgaacc aaagagtgcc aatattccct ggttatgcac cctccaagcg 120 gtgggagaagaattcggccc agccagagtg catgtacctt tttctctctc acacttgaag 180 caaattaaaatagacctagg taaattctca gatagccctg atggctatat tgatgtttta 240 caaggattaggacaatcctt tgatctgaca tggagagata taatattact gctaaatcag 300 acgctaacctcaaatgagag aagtgctgcc ataactggag cccgagagtt tggcaatctc 360 tggtatctcagtcaggtcaa tgataggatg acaacggagg aaagagaacg attccccaca 420 gggcagcaggcagttcccag tgtagctcct cattgggaca cagaatcaga acatggagat 480 tggtgccgcagacattta 498 31 166 PRT MSRV 31 Leu Leu Pro Gln Leu Ile Arg Thr Pro LeuSer Thr Gln Thr Val Gln 1 5 10 15 Lys Asp Ile Asp Lys Gly Val Asn AsnGlu Pro Lys Ser Ala Asn Ile 20 25 30 Pro Trp Leu Cys Thr Leu Gln Ala ValGly Glu Glu Phe Gly Pro Ala 35 40 45 Arg Val His Val Pro Phe Ser Leu SerHis Leu Lys Gln Ile Lys Ile 50 55 60 Asp Leu Gly Lys Phe Ser Asp Ser ProAsp Gly Tyr Ile Asp Val Leu 65 70 75 80 Gln Gly Leu Gly Gln Ser Phe AspLeu Thr Trp Arg Asp Ile Ile Leu 85 90 95 Leu Leu Asn Gln Thr Leu Thr SerAsn Glu Arg Ser Ala Ala Ile Thr 100 105 110 Gly Ala Arg Glu Phe Gly AsnLeu Trp Tyr Leu Ser Gln Val Asn Asp 115 120 125 Arg Met Thr Thr Glu GluArg Glu Arg Phe Pro Thr Gly Gln Gln Ala 130 135 140 Val Pro Ser Val AlaPro His Trp Asp Thr Glu Ser Glu His Gly Asp 145 150 155 160 Trp Cys ArgArg His Leu 165 32 286 DNA MSRV 32 cacaggggaa aggaagaaaa tcctactgcctttctggaga gactaaggga ggcattgagg 60 aagcatacca ggcaagtgga cattggaggctctggaaaag ggaaaagttg ggaaaagtat 120 atgtctaata gggcttgctt ccagtgtggtctacaaggac actttaaaaa agattgtcca 180 atagaaataa gccaccacct cgtccatgccccttatgtca agggaatcac tggaaggccc 240 actgccccag gggatgaagg tcctctgagtcagaagccac taacca 286 33 95 PRT MSRV 33 His Arg Gly Lys Glu Glu Asn ProThr Ala Phe Leu Glu Arg Leu Arg 1 5 10 15 Glu Ala Leu Arg Lys His ThrArg Gln Val Asp Ile Gly Gly Ser Gly 20 25 30 Lys Gly Lys Ser Trp Glu LysTyr Met Ser Asn Arg Ala Cys Phe Gln 35 40 45 Cys Gly Leu Gln Gly His PheLys Lys Asp Cys Pro Ile Glu Ile Ser 50 55 60 His His Leu Val His Ala ProTyr Val Lys Gly Ile Thr Gly Arg Pro 65 70 75 80 Thr Ala Pro Gly Asp GluGly Pro Leu Ser Gln Lys Pro Leu Thr 85 90 95 34 20 DNA ArtificialSequence primer 34 tgaagagagt caaaagcagc 20

What is claimed is:
 1. Polypeptide capable of reacting specifically withthe antibodies of patients suffering from multiple sclerosis (MS) andwhose peptide sequence comprises at least one sequence chosen from SEQID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12 to SEQID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 19, said polypeptide having nomore than 20 amino acids.
 2. Polypeptide of claim 1, said polypeptidehaving no more than 15 amino acids.
 3. Reagent for the detection ofmultiple sclerosis in a patient and/or the monitoring of a patientsuffering from multiple sclerosis, characterized in that it comprises atleast one polypeptide according to claim 1, said polypeptide beingoptionally labeled.
 4. Reagent according to claim 3, characterized inthat it comprises at least two different polypeptides.
 5. Polynucleotidewhose nucleotide sequence encodes a polypeptide according to claim
 1. 6.Polypeptide capable of reacting with the antibodies of patientssuffering from multiple sclerosis (MS) and whose peptide sequenceconsists of a sequence selected from SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 8, SEQ ID NO: 12 to SEQ ID NO: 14, SEQ ID NO: 16 andSEQ ID NO:
 19. 7. Polypeptide capable of reacting with at least oneantibody in at least one biological fluid from a patient in whom theMSRV-1 viral sequences have been detected and whose peptide sequencecomprises at least one sequence chosen from SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 4, said polypeptide having no more than 20 amino acids. 8.Polypeptide of claim 7, said polypeptide having no more than 15 aminoacids.
 9. Reagent for the detection of an MSRV-1 virus infection,characterized in that it comprises at least one polypeptide according toclaim 7, said polypeptide being optionally labeled.
 10. Polypeptidecapable of reacting with at least one antibody in at least onebiological fluid from a patient in whom MSRV-1 viral sequences have beendetected and whose peptide sequence consists of a sequence chosen fromSEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO:
 4. 11. Kit for the detection ofmultiple sclerosis in a patient and/or the monitoring of a patientsuffering from multiple sclerosis, comprising: a reagent for thedetection of multiple sclerosis in a patient and/or the monitoring of apatient suffering from multiple sclerosis, the reagent comprising atleast one polypeptide capable of reacting specifically with theantibodies of patients suffering from multiple sclerosis (MS) and whosepeptide sequence comprises at least one sequence chosen from SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 8; SEQ ID NO: 12 to SEQ ID NO:14, SEQ ID NO: 16 and SEQ ID NO: 19; the polypeptide having no more than20 amino acids; and the polypeptide being optionally labeled wherein thereagent is supported on a support which is immunologically compatiblewith said reagent.
 12. Kit for the detection of an MSRV-1 virusinfection, comprising: a reagent for the detection of an MSRV-1 virusinfection; the reagent comprising at least one polypeptide capable ofreacting with at least one biological fluid from a patient in whom theMSRV-1 viral sequences have been detected and whose peptide sequencecomprises at least one sequence chosen from SEQ ID NO: 1 to SEQ ID NO:4; the polypeptide having no more than 20 amino acids; and thepolypeptide being optionally labeled; wherein the reagent is supportedon a support which is immunologically compatible with said reagent. 13.Method of binding, in a biological sample, antibodies which arecharacteristic of and/or specific to multiple sclerosis, comprising:bringing the sample into contact with a reagent for the detection ofmultiple sclerosis in a patient and/or the monitoring of a patientsuffering from multiple sclerosis, the reagent comprising at least onepolypeptide capable of reacting specifically with the antibodies ofpatients suffering from multiple sclerosis (MS) and whose peptidesequence comprises at least one sequence chosen from SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12 to SEQ ID NO: 14,SEQ ID NO: 16 and SEQ ID NO: 19; the polypeptide having no more than 20amino acids; and the polypeptide being optionally labeled; optionallydetecting the presence of an immune complex; and optionally separatingthe detected immune complex.
 14. Method according to claim 13,characterized in that the biological sample is chosen from serum,cerebrospinal fluid and urine.
 15. Method of binding, in a biologicalsample, antibodies directed against the MSRV-1 virus, comprising:bringing the sample into contact with a reagent for the detection of anMSRV-1 virus infection; the reagent comprising at least one polypeptidecapable of reacting with at least one biological fluid from a patient inwhom the MSRV-1 viral sequences have been detected and whose peptidesequence comprises at least one sequence chosen from SEQ ID NO: 1 to SEQID NO: 4; the polypeptide having no more than 20 amino acids; and thepolypeptide being optionally labeled; optionally detecting the presenceof an immune complex; and optionally separating the detected immunecomplex.
 16. Kit for the detection of multiple sclerosis in a patientand/or the monitoring of a patient suffering from multiple sclerosis,comprising: a reagent for the detection of multiple sclerosis in apatient and/or the monitoring of a patient suffering from multiplesclerosis; the reagent comprising at least one polypeptide capable ofreacting specifically with the antibodies of patients suffering frommultiple sclerosis (MS) and whose peptide sequence comprises at leastone sequence chosen from SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQID NO: 8, SEQ ID NO: 12 to SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO:19; the polypeptide having no more than 20 amino acids; and thepolypeptide being optionally labeled; wherein the reagent is supportedon a support that does not react with the reagent.
 17. Kit for thedetection of an MSRV-1 virus infection, comprising: a reagent for thedetection of an MSRV-1 virus infection; the reagent comprising at leastone polypeptide capable of reacting with at least one biological fluidfrom a patient in whom the MSRV-1 viral sequences have been detected andwhose peptide sequence comprises at least one sequence chosen from SEQID NO: 1 to SEQ ID NO: 4; the polypeptide having no more than 20 aminoacids; and the polypeptide being optionally labeled; wherein the reagentis supported on a support that does not react with the reagent.